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cancer > 9. p53 > Flashcards

9. p53 Flashcards

1
Q

name the major regulator of p53 and what is the human version

A

MDM2

HDM2

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2
Q

what was MDM2 gene clones as

A

a gene that conferred a growth advantage - bits of mice chromosome put into mice cells to see which chromosomal fragment enhanced cell growth

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3
Q

when MDM2 is over expressed what type of signal is this and describe the results of two tumourgenic assays, and what does this show?

A

pro-proliferative
>induces focus formation in vitro and tumours in nude mice
>MDM2 is oncogenic

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4
Q

immunoprecipitation of MDM2 showed what

A

it binds p53

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5
Q

what regions of p53 does MDM2 bind and what affect does this have?

A

MDM2 binds TA domains on p53
>MDM2 is a large proteins and so prevents other proteins binding TA domain
>p53 is no longer a transcriptional regulator when bound MDM2

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6
Q

in addition to MDM2 regulating p53 by masking the transactivation domain what else can it do?

A

cause p53 to be lost from cells

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7
Q

when the proteasome is inhibited, what is observed in cells with MDM2 and p53? and what does this suggest?

A

p53 is not lost from cells

>suggests that MDM2 activates proteolysis of p53.

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8
Q

when MDM2 binds p53, what does it act as? and what does this cause?

A

a ubiquitin ligase. this is a signal for degradation by the proteasome.

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9
Q

describe the MDM2/p53 feedback loop

A

MDM2 is a target genes of p53
>negative feedback loop that limits p53 function
>loop senses how much damage there is, if there is lots of damage p53 will remain active, if the damage is gone, MDM2 will switch off p53
>important to keep a balance between the two

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10
Q

what binds to MDM2 and inhibits its ubiquitin ligase activity? and what affect does this have?

A

Arf

>this stabilises p53

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11
Q

why might MDM2 levels be higher when Arf is present?

A

Arf stabilises and inhibits MDM2

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12
Q

describe the genomic organisation of Arf

A

Arf and p16 superimpose each other
>they share some DNA sequence but there is no sequence homology as they have different reading frames
>Arf and p16 have different promoters

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13
Q

why would you put two key cell cycle regulator tumour suppressers in the same piece of DNA that could be knocked out?

A

there is no explanation for this

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14
Q

what can activate transcription of p16 and ARF?

A

activated ras

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15
Q

what else can bind Arf?

A

E2F and E1A

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16
Q

what is the Arf p16 locus critical for?

A

supressing oncogenic activation
p16 - cell cycle arrest
Arf - p53 response

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17
Q

name the disease that is associated with mutations in ATM? what are some symptoms?

A

Ataxia Telangiectasia
>cancer prone disease
>autosomal recessive

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18
Q

describe the ATM body plan

A

when it was isolated the majority of it didn’t look like anything that had ever been cloned
>apart from C terminus which has a PI3Kinase domain

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19
Q

what are the three family members of ATM?

A

ATM, ATR and ATX

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20
Q

what type of protein is ATM?

A

serine threonine kinase

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21
Q

UV radiation is largely what type of DNA damage?

A

single strand breaks

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22
Q

ionising radiation is largely what type of DNA damage?

A

double strand breaks

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23
Q

when ATM is KO and DNA is damaged what happens and what does this show?

A

> patients don’t respond to ionising damage
but they do respond to UV damage
ATM is part of dsDNA response

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24
Q

when ATR is KO and DNA is damaged what happens and what does this how? why can ATR not be truly KO?

A

> patients don’t respond to UV radiation
patients do respond to ionising damage
ATR is part of the ssDNA response
it is also part of DNA replication and this is needed to make viable cells, need ATR null cells in sense of DNA damage response

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25
Q

where is ATM found?

A

in close association with chromatin in an inactive homodimer

26
Q

when DNA is damaged what does this lead to and how does this affect ATR?

A

> DNA damage leads to changes in chromatin structure.
this leads to changes in the ATR homodimer which leads to trans-autophosphorylation
dimer dissociates into active monomers

27
Q

what can also occur in terms of ATM when dsDNA breaks occur?

A

M, R and N are three high affinity proteins for end of DNA
>once bound to DNA they undergo conformational changes which allow them to bind ATM dimer
>ATM dimer trans-autophosphorylates to form active monomers.

28
Q

how can ATM be activated by oxidative agents?

A

> dimer associated through non-covalent interactions
cysteine in ATM very sensitive to oxidative damage
disulphide bonds form as ATM is acted on by oxidative agents
formation of disulphide associated with P of monomers and activation

29
Q

in additions to mutations in p53, what other mutations is characteristic of people with Li-Fraumeni Syndrome?

A

Chk2 - serine/threonine kinase

30
Q

what happens when Chk2 is mutated in yeast?

A

they become hypersensitive to damage

31
Q

when cells are damaged with ionising radiation what activation does this lead to?

A

ATM

32
Q

what does ATM P? and what does this affect?

A

p53 and Cdk2
Chk2 then P p53
this affects the N terminal region of p32 - the region that binds MDM2

33
Q

why does p53 need to be P twice? and what does this result in?

A

in needs to be P twice so than MDM2 cannot bind

>this results in the stabilisation of p53 and so increased response

34
Q

what three things might influence if there is arrest or apoptosis?

A
  • strength of signal
  • p53 promoters binding affinity
  • modifications
  • other regulators
35
Q

how will the strength of the signal determine where there is arrest or apoptosis?

A

lost of damage = lots of p53

more damage = more death

36
Q

how will the p53 promoter binding affinity determine where there is arrest or apoptosis?

A

p53 promoters are degenerate and so will have different binding affinity
>higher affinity will bind first - arrest genes
>high levels of p53 will able bind apoptotic genes

37
Q

how will the modifications determine where there is arrest or apoptosis?

A

p53 is subject to post-translational modifications e.g. phosphorylation, sumoylation, acetylation - these affect function and localisation

38
Q

p53 can be acetylated on lysines, what affect does this have?

A

this has different effects on the subset of promoters that it binds, this can additionally be affected with combinations of P and sumoylations
>different combos of PTM regulate which promoter p53 binds and with what affinity

39
Q

what proteins can bind p53 and modify its death/arrest response? and what affect does this have?

A

ASPP2

>causes p53 to activate apoptosis genes

40
Q

when looking at a flow cytometry plot, what can be assumed about cells that are in sub-G1 population?

A

they are undergoing apoptosis - cells are breaking down their DNA in the apoptotic process

41
Q

what have people developed in terms of computer programmes?

A

programmes that can predict the p53 outcomes (arrest/response) depending on the DNA damage - in order to see what will happen in different DNA damage settings

42
Q

what protein de-P p53 so that MDM2 can bind?

A

Wip1

43
Q

when Akt P MDM2 what affect does this have?

A

make its better at degrading p53

44
Q

what were the outputs for long and short DNA damage from this computer simulation? and what does this suggest about p52 signalling?

A

> short DNA damage leads to spikes in ATM activity
long DNA damage leads to spikes in ATM followed by sustained DNA activity - this leads to apoptosis
this suggests that p53 signalling is digital - if the damage persists for long time they you get sustained signalling

45
Q

what was shown in cells and how did this differ from the computer programme?

A

when damage occurs p53 levels rise, they decline and then come back more/less strongly than the first time.
>p53 cycling conferred with the model but the response is not uniform through cell population and so the model needs to be refined

46
Q

name three other p53 family members, what do they all have? what is conserved and what does this suggest?

A

p53
p63
p73
>all have TA domain, DNA binding domains and tetramer formation domain
>conserved DNA binding site suggests they bind the same promoters

47
Q

describe the p73 genome organisation, how does this compare to p63? and what proteins are produced?

A

there are 2 promoters
one at the 5’ end
and one between exons 3 and 4
transcription from this results in N terminal truncation - no TA domain
>the same occurs in p63
>these are dominant negative TFs (bind DNA don’t activate transcription)

48
Q

the 5’ promoter binds lots of TFs, what 2 TFs bind the promoter found between exons 3 and 4 in p73?

A

p53 and truncated p73

49
Q

what is the purpose of these truncated p73 and p63?

A

to dampen down the p53 response - they inhibit p53 dependent transcription

50
Q

p73 can also be activated by ATM following DNA damage. what does this result in?

A

some p73 binds P2 and produce a transcription factor that doesn’t activate transcription
>they compete for binding sites of target promoters with transcriptionally activating proteins like p53

51
Q

when are the truncated proteins generated and what implication does this have?

A

they are generated after p53 has risen
>this means that p53 has had some affect before they are transcribed
>they can then controlling the levels of response in DNA damaged by blocking promoters.

52
Q

how do p63 and p73 function? and what happens if truncated protein is incorporated? and what does this mean?

A

as tetramers
>heterodimer will have some, but reduced activity - some wont have TA
>p53 signal is reduced

53
Q

how might truncated p63 affect the p53 response?

A
  • determine how long and how you respond to DNA damage
  • DNA binding sequence not identical to p53 and so will bind subset of promoters with higher affinity
  • this might attribute to the type of p53 response obtained
54
Q

how often are p73 and p63 mutated in cancer and what does this suggest?

A

not very often - they are not critical factors

they just modulate the p53 response

55
Q

name two therapeutic strategies used in terms of p53

A
  • activation of WT protein

- reactivation of mutant protein

56
Q

what type of p53 are hard to drug? and which are more drugable?

A

> one with mutations in the DBD - hard to give back binding specificity
mutations that cause conformational changes and these are more drugable by trying to stabilise the active folded p53.

57
Q

50% of tumours have mutations in p53, this means that half still have active p53. what is seen in some tumours with active p53? and what implication does this have?

A

upregulation of MDM2

>one way to activate p53 in tumours is to stop MDM2 binding it

58
Q

name an MDM2 inhibitor and what does it do?

A

nutlin
>small molecule that sits of MDM2 where the TA helix would site and blocks interaction with p53
>it competes for binding well
>re-establishes p53 response in cells with WT p53

59
Q

what is an advantage of nutlin over chemo?

A

nutlin is not cytotoxic

60
Q

what can be used to reactive mutated p53?

A

there are small molecules specific for mutations. which stabilise mutant form and allow protein to properly fold
>transcription based assay shows that these regain function

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